Urinary Profiles Research Articles

Comprehensive characterization of Epimedium-Rhizoma drynariae herb pair in rat plasma, urine, and feces metabolic profiles by UHPLC-Q-Orbitrap HRMS combined with diagnostic extraction strategy and multicomponent pharmacokinetic study by UHPLC-MS/MS.

Epimedium-Rhizoma drynariae (EP-RD) was a well-known herb commonly used to treat bone diseases in traditional Chinese medicine. Nevertheless, there was incomplete pharmacokinetic behavior, metabolic conversion and chemical characterization of EP-RD in vivo. Therefore, this study aimed to establish metabolic profiles combined with multicomponent pharmacokinetics to reveal the in vivo behavior of EP-RD. Firstly, the diagnostic product ions (DPIs) and neutral losses (NLs) filtering strategy combined with UHPLC-Q-Orbitrap HRMS for the in vitro chemical composition of EP-RD and metabolic profiles of plasma, urine, and feces after oral administration of EP-RD to rats were proposed to comprehensively characterize the 47 chemical compounds and the 97 exogenous in vivo (35 prototypes and 62 metabolites), and possible biotransformation pathways of EP-RD were proposed, which included phase I reactions such as hydrolysis, hydrogenation, dehydrogenation, hydroxylation, dehydroxylation, isomerization, and demethylation and phase II reactions such as glucuronidation, acetylation, methylation, and sulfation. Moreover, a UHPLC-MS/MS quantitative approach was established for the pharmacokinetic analysis of seven active components: magnoflorine, epimedin A, epimedin B, epimedin C, icariin, baohuoside II, and icariin II. Results indicated that the established method was reliably used for the quantitative study of plasma active ingredients after oral administration of EP-RD in rats. Compared to oral EP alone, the increase in area under curves and maximum plasma drug concentration (P < 0.05). This study increased the understanding of the material basis and biotransformation profiles of EP-RD in vivo, which was of great significance in exploring the pharmacological effects of EP-RD.

The acute phase reactant orosomucoid-2 directly promotes rheumatoid inflammation

Acute phase proteins involved in chronic inflammatory diseases have not been systematically analyzed. Here, global proteome profiling of serum and urine revealed that orosomucoid-2 (ORM2), an acute phase reactant, was differentially expressed in rheumatoid arthritis (RA) patients and showed the highest fold change. Therefore, we questioned the extent to which ORM2, which is produced mainly in the liver, actively participates in rheumatoid inflammation. Surprisingly, ORM2 expression was upregulated in the synovial fluids and synovial membranes of RA patients. The major cell types producing ORM2 were synovial macrophages and fibroblast-like synoviocytes (FLSs) from RA patients. Recombinant ORM2 robustly increased IL-6, TNF-α, CXCL8 (IL-8), and CCL2 production by RA macrophages and FLSs via the NF-κB and p38 MAPK pathways. Interestingly, glycophorin C, a membrane protein for determining erythrocyte shape, was the receptor for ORM2. Intra-articular injection of ORM2 increased the severity of arthritis in mice and accelerated the infiltration of macrophages into the affected joints. Moreover, circulating ORM2 levels correlated with RA activity and radiographic progression. In conclusion, the acute phase protein ORM2 can directly increase the production of proinflammatory mediators and promote chronic arthritis in mice, suggesting that ORM2 could be a new therapeutic target for RA.

Characterization of iso-LSD metabolism using human liver microsomes in comparison to LSD and its applicability as urinary biomarker for LSD consumption.

Urinalysis of lysergic acid diethylamide (LSD) poses a challenge due to its rapid metabolism, resulting in little to no LSD detectable in urine. Instead, its primary metabolite, 2-oxo-3-hydroxy-LSD, is predominantly detected. In this study, we observed several urine profiles with iso-LSD detected together with 2-oxo-3-hydroxy-LSD. Iso-LSD is derived from illicit preparation of LSD as a major contaminant, and it was detected at higher abundance than LSD and 2-oxo-3-hydroxy-LSD in certain urine samples. Therefore, the metabolism of iso-LSD and its potential as a viable urinary biomarker for confirming LSD consumption is of interest. For metabolism studies, LSD and iso-LSD were incubated in human liver microsomes (HLMs) at 0 min, 60 min and 120 min to characterize their metabolites using LC-QTOF-MS. For urinary analysis, 500 µL of urine samples underwent enzymatic hydrolysis and clean-up using supported-liquid extraction (SLE) prior to analysis by LC-QTOF-MS. From HLM incubation study of LSD, the metabolites detected were dihydroxy-LSD, 2-oxo-LSD, N-desmethyl-LSD (nor-LSD) and 2-oxo-3-hydroxy-LSD with LSD levels decreasing significantly throughout all time points, consistent with the existing literatures. For HLM study of iso-LSD, metabolites eluting at retention times after the corresponding metabolites of LSD were detected, with iso-LSD levels showing only a slight decrease throughout all time points, due to a slower metabolism of iso-LSD compared to LSD. These findings corroborate with the urinalysis of 24 authentic urine samples, where iso-LSD with 2-oxo-3-hydroxy-LSD was detected in the absence of LSD. Based on our findings, iso-LSD is commonly detected in urine (18 out of 24 samples) sometimes with traces of possible 2-oxo-3-hydroxy-iso-LSD. The slower metabolism and high detection rate in urine make iso-LSD a viable urinary biomarker for confirming LSD consumption, especially in the absence of LSD and/or 2-oxo-3-hydroxy-LSD.

Abstract 5039: Preanalytical workflow enabling cfDNA analysis from urine samples

Abstract Introduction: Urine has become an important source of information in the liquid biopsy field. In contrast to blood, specifications for the collection, storage, transport and processing of urine intended for molecular examination are not widely established. Preanalytical specifications were published for urine cell-free DNA (cfDNA) only recently (CEN/TS 17811:2022). In this study, we investigated post-collection changes to cfDNA profiles in urine samples and present the performance of an optimized preanalytical workflow for cfDNA analysis. Methods: Urine from apparently healthy, consented female individuals was spiked with processed cell-free male urine or with a cfDNA standard containing PIK3CA mutations (SensID). The urine was stabilized with a urine collection/stabilization solution under development at PreAnalytiX or was left unstabilized. Urine samples were stored for varying durations at different temperatures in a time course experiment to simulate different urine storage conditions. After storage, urine samples were centrifuged to remove cells and cfDNA was isolated from the supernatant via the QIAsymphony® platform (QIAGEN). Autosomal and male-specific targets were quantified using the Rotor-Gene® Q instrument and the therascreen® PIK3CA qPCR Kit (both QIAGEN) or the QIAcuity® Digital PCR System and a dPCR LNA PIK3CA Mutation Assay (both QIAGEN). Fragment size distribution was determined by TapeStation® Cell-free DNA ScreenTape® (Agilent Technologies). Results: Analysis of cfDNA profiles in unstabilized urine stored for varying durations and temperatures highlighted changes which create artificial cfDNA profiles. The yield of cfDNA drastically decreased over time. Furthermore, the size distribution of cfDNA isolated from unstabilized urine was impacted by release of gDNA as well as DNA degradation. Urine stabilization minimized cfDNA degradation and gDNA release and allowed isolation of cfDNA that was analyzed with qPCR and dPCR even after urine storage. Analysis of isolated cfDNA revealed positive detection of PIK3CA mutations in spiked urine samples. Conclusions: Changes in post-collection cfDNA profile can hinder downstream analysis, resulting in artificial or failed outcomes. Urine stabilization with a collection/stabilization solution under development at PreAnalytiX minimized DNA degradation and gDNA release. It enabled urine storage, allowed analysis of urine cfDNA and target detection by qPCR and dPCR. Citation Format: Daniela Mancarella, Julia Rutsch, Lisa Erkelenz, Moritz Meyer, Laura Sofie Witthaus, Moritz Rath, Thorsten Voss. Preanalytical workflow enabling cfDNA analysis from urine samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5039.

Preliminary study of urine analysis in pregnant cows: insights for improved herd management

ABSTRACT This study aimed to establish a standard urine profile for pregnant cows, commonly seen patients in veterinary clinics. Urine samples from 15 pregnant cows (6 Belgian Blue and 9 Holstein) of various ages were analysed, all showing no clinical signs of urinary tract disease. Samples were collected during preventive check-ups, either by spontaneous micturition or catheterisation. Macroscopic evaluation and urine dipstick tests revealed a mean pH of 8.17 ± 0.49. Moderate glycosuria and proteinuria were detected in two animals. No urobilinogen, ketone bodies, haemoglobin, or traces of blood were found. Laboratory urinalysis provided values (with 95% confidence intervals): osmolarity 493–1110.00 mOsm/l, sodium 52.00–122.00 mmol/l, calcium 1.28–3.98 mmol/l, potassium 76–144.00 mmol/l, magnesium 5.00–8.00 mmol/l, chlorine 40.00–80.00 mmol/l, protein 35.00–104.00 mg/l, glucose 0.40–1.27 mmol/l, creatinine 34.00–76.00 mmol/l, and urea 681.00–2086 mmol/l. These findings present the initial descriptive values of urine biochemical parameters in pregnant cows, emphasising their significance in assessing animal health. This research underscores its relevance by providing fundamental insights into animal health, offering a pathway to enhance agricultural sustainability and address food security, echoing the United Nations’ Sustainable Development Goals 1, 2, 8 and 12.

Determining Kidney Damage Risk Factors Associated with Urine Paraquat Levels and Biochemical Urine Profile of Farmers in Banjarbaru, South Kalimantan, Indonesia

Abstract Aim: Kidney damage from paraquat is influenced by several factors, including the duration of work, the span of exposure from the pesticide spraying, and the nutritional status of the farmers. This damage can be indicated by the high levels of paraquat and changes of the biochemical status in the urine. The present study aimed to analyze the relationship between the length of work, duration of spraying, and the nutritional status obtained from the measurement of body mass index (BMI) values, with the paraquat levels and biochemical status of the farmer’s urine samples. Materials and Methods: This study was a cross-sectional study and was conducted in September 2022 in North Landasan Ulin Village, Banjarbaru City, South Kalimantan, Indonesia. The length of work and spraying duration were obtained from the direct interviews, while the BMI values were calculated by dividing the body weight (kg) by the body’s height (m). Analysis of the paraquat levels in the urine was measured using a spectrophotometer, while the urine’s biochemical status was obtained using the dipstick colorimetric method. Statistical analysis used: the relationship between each variable is analyzed using the Spearman rank correlation test. Results: Results from the study show that the length of work, duration of spraying, and BMI were moderately associated with an increase in the urinary paraquat levels (correlation coefficient [Rs] 0.26–0.50) (P &lt; 0.05); although the previous factors did not appear to correlate with any changes in the urine’s biochemical status. Conclusion: In summary, prolonged exposure to the spray in combination with the farmers’ long work hours, and also factoring in the abnormal BMI values, resulted in an increase of paraquat levels in the urine.

Metabolic profiling in tissues and urine of patients with prostatic lesions and the diagnostic value of urine extracellular vesicles metabolites in prostate cancer

BackgroundProstate cancer (PCa) lacks convenient and highly specific diagnostic markers. Although the value of extracellular vesicles (EV) in oncology is widely recognized, the diagnostic value of EV metabolites requires further exploration. This study aimed to explore the diagnostic value of urine EV (u-EV) metabolomics in PCa. MethodsWe first detected metabolites in paired tissues cells (cells), tissue EV (t-EVs), u-EVs, and urine samples in cohort 1 (8 PCa vs. 5 benign prostatic hypertrophy, BPH) to prob the feasibility of EV metabolites as diagnostic markers. We then analyzed the value of u-EVs as markers for PCa diagnosis and typing in the expanded sample cohort (60 PCa vs. 40 BPH). ResultsU-EV metabolites were more consistent with those in tissue-derived samples (cells and t-EVs) than those in urine, and more differential metabolites between BPH and PCa were identified in u-EV. Subsequently, we used a random forest model to construct a panel of six metabolites for PCa, which showed an area under the curve (AUC) of 0.833 in training cohort and 0.844 in validation cohort. We also found significantly differentially expressed metabolites between PCa subtypes (Gleason ≤ 7 vs. Gleason > 7 and localized vs. metastasis), demonstrating the value of EV metabolites in PCa typing and prognostic assessment. ConclusionMetabolomic analysis of u-EVs is a promising source of noninvasive markers for PCa diagnosis.

Lipidomic profiles in serum and urine in children with steroid sensitive nephrotic syndrome

BackgroundSteroid-sensitive nephrotic syndrome (SSNS) accounts for approximately 80% of cases of nephrotic syndrome. The involvement of aberrant lipid metabolism in early SSNS is poorly understood, warranting further investigation. This study aimed to explore alterations in lipid metabolism associated with SSNS pathogenesis. MethodsA screening cohort containing serum (50 SSNS, 37 controls) and urine samples (27 SSNS, 26 controls) was analyzed by untargeted lipidomic profiling using UHPLC-QTOF-MS. Then, a validation cohort (20 SSNS, 56 controls) underwent further analysis to check the potential clinical application by ROC curve analysis. ResultsLipidomic profiling of serum and urine samples revealed significant lipid alterations in SSNS patients, with the alterations in the serum samples being more significant. An elevated concentration of PE and PG and downregulated concentration of FA were observed in SSNS serum. A total of 38 dysregulated lipids and 5 lipid metabolic pathways were identified in the serum samples in SSNS patients. Validation in the second cohort confirmed differential regulation of nine kinds of lipids, including 5 up-regulated substances [SM d33:2 (m/z = 686.5361), SHexCer d34:1 (m/z = 779.521), PI 20:4_22:4 (m/z = 934.5558), Cer_NS d18:1_23:0 (m/z = 635.6216), and GM3 d36:1 (m/z = 1180.7431)], as well as 4 down-regulated substances: [CE 18:1 (m/z = 650.601), PE 38:6 (m/z = 763.5205), PC 17:0_20:4 (m/z = 795.5868) and EtherPC 16:2e_20:4 (m/z = 763.5498)]. ConclusionsUntargeted lipidomic analysis successfully identified specific lipid class changes in patients with SSNS, providing a deeper understanding of lipid alterations and underlying mechanisms associated with SSNS.

P442 Renal lithogenic risk in patients with Inflammatory Bowel Diseases correlates with microbiota composition, urinary profile and dietary intake

Abstract Background Nephrolithiasis (NL) is a common extra-intestinal complication of inflammatory bowel diseases (IBD), often related to enteric hyperoxaluria. Growing evidence highlights the association between NL and gut microbiota, but no data are available on the urinary microbiota in IBD patients. Our study aims to analyse the relationship between diet, urinary lithogenic profile, and gut and urinary microbiota in patients with IBD. Methods We prospectively enrolled consecutive adult patients with IBD. All patients underwent abdominal ultrasound, blood tests and 24h-urine collection for lithogenic profile analysis. Moreover, a nutritional evaluation was performed, and all patients underwent bioimpedance measurement. Urinary and gut microbiota were analysed through 16S rRNA sequencing. Results 60 patients were enrolled: 32 with Crohn’s disease (CD) and 28 with ulcerative colitis (UC). NL was more frequent in CD than in UC (28% vs 14%). In patients with NL, gut microbiota analysis showed a lower relative abundance of Agathobacter (p&amp;lt;0.05) and a reduction in alpha diversity (p&amp;lt;0.05) [figure 1]. No statistically significant difference was found in urinary microbiota composition comparing patients with the presence or absence of NL. Protein (g/die) and phosphorus (mg/die) dietary intake were significantly associated with oxaluria (mg/L) (p=0.032 and p=0.007, respectively). At the same time, protein and calcium (mg/day) intake were significantly associated with NL (p= 0.018 and p= 0.023, respectively). Oxaluria and calcium oxalate relative saturation ratio (RSRCaOx) were significantly higher in CD compared to UC [Table 1]. Conclusion Patients with IBD and NL have distinctive gut microbiota features. CD patients showed a higher prevalence of NL and oxaluria levels than UC. In addition, NL occurrence and oxaluria correlated with some nutritional parameters. Further studies are needed to identify patients with IBD at greater risk of NL by connecting the parameters of the gut microbiota, urinary profile and dietary intake to act on modifiable factors aiming to reduce this risk.

Detection of FG-4592 and metabolites in equine plasma, urine and hair following oral administration.

FG-4592 is a hypoxia-inducible factor inhibitor that has been approved for therapeutic use in some countries. This class of compounds can increase the oxygen carrying capacity of the blood and thus have the potential to be used as performance enhancing agents in sports. The purpose of this study was to investigate the detection of FG-4592 and metabolites in equine plasma and mane hair following a multiple dose oral administration to two Thoroughbred racehorses, to identify the best analytical targets for doping control laboratories. Urine samples were also analysed, and the results compared to previously published urine data. Liquid chromatography-high resolution mass spectrometry was used for metabolite identification in urine and plasma. Liquid chromatography-tandem mass spectrometry was used for full sample analysis of urine, plasma and hair samples and generation of urine and plasma profiles. FG-4592 and a mono-hydroxylated metabolite were detected in plasma. FG-4592 was detected with the greatest abundance and gave the longest duration of detection, up to 312 h post-administration, and would be the recommended target in routine doping samples. FG-4592 was detected in all mane hair samples collected post-administration, up to 166 days following the final dose, showing extended detection can be achieved with this matrix. To the best of the authors' knowledge, this is the first report of FG-4592 and metabolites in equine plasma and hair samples. Urine results were consistent with the previously published data, with FG-4592 offering the best target for detection and longest detection periods.

Population Pharmacokinetic Analysis of Atomoxetine and its Metabolites in Children and Adolescents with Attention-Deficit/Hyperactivity Disorder.

Atomoxetine (ATX) is a non-stimulant used to treat attention-deficit/hyperactivity disorder (ADHD) and systemic exposure is highly variable due to polymorphic cytochrome P450 2D6 (CYP2D6) activity. The objective of this study was to characterize the time course of ATX and metabolites (4-hydroxyatomoxetine (4-OH); N-desmethylatomoxetine (NDA); and 2-carboxymethylatomoxetine (2-COOH)) exposure following oral ATX dosing in children with ADHD to support individualized dosing. A nonlinear mixed-effect modeling approach was used to analyze ATX, 4-OH, and NDA plasma and urine, and 2-COOH urine profiles obtained over 24-72 hours from children with ADHD (n = 23) following a single oral ATX dose. Demographics and CYP2D6 activity score (AS) were evaluated as covariates. Simulations were performed to explore the ATX dosing in subjects with various CYP2D6 AS. A simultaneous pharmacokinetic modeling approach was used in which a model for ATX, 4-OH, and NDA in plasma and urine, and 2-COOH in urine was developed. Plasma ATX, 4-OH, and NDA were modeled using two-compartment models with first-order elimination. CYP2D6 AS was a significant determinant of ATX apparent oral clearance (CL/F), fraction metabolized to 4-OH, and systemic exposure of NDA. CL/F of ATX varied almost 7-fold across the CYP2D6 AS groups: AS 2: 20.02 L/hour; AS 1: 19.00 L/hour; AS 0.5: 7.47 L/hour; and AS 0: 3.10 L/hour. The developed model closely captures observed ATX, 4-OH, and NDA plasma and urine, and 2-COOH urine profiles. Application of the model shows the potential for AS-based dosing recommendations for improved individualized dosing.

Metabolomic analysis of the serum and urine of rats exposed to diazinon, dimethoate, and cypermethrin alone or in combination

BackgroundMultiple pesticides are often used in combination for plant protection and public health. Therefore, it is important to analyze the physiological changes induced by multiple pesticides exposure. The objective of this study was to investigate the combined toxicity of the widely-used organophosphorus and pyrethroid pesticides diazinon, dimethoate, and cypermethrin.MethodsMale Wistar rats were administrated by gavage once daily with the three pesticides individual or in combination for consecutive 28 days. The metabolic components of serum and urine samples were detected by using 1H nuclear magnetic resonance (NMR)-based metabolomics method. Histopathological examination of liver and kidneys and serum biochemical determination were also carried out.ResultsThe results showed that after the 28-day subacute exposure, serum glutamic transaminase and albumin were significantly increased and blood urea nitrogen was significantly decreased in the rats exposed to the mixture of the pesticides compared with the control rats, suggesting that the co-exposure impaired liver and kidney function. Metabolomics analysis indicated that the indicators 14 metabolites were statistically significant altered in the rats after the exposure of the pesticides. The increase in 3-hydroxybutyric acid in urine or decrease of lactate and N-acetyl-L-cysteine in serum could be a potentially sensitive biomarker of the subchronic combined effects of the three insecticides. The reduction level of 2-oxoglutarate and creatinine in urine may be indicative of dysfunction of liver and kidneys.ConclusionIn summary, the exposure of rats to pesticides diazinon, dimethoate, and cypermethrin could cause disorder of lipid and amino acid metabolism, induction of oxidative stress, and dysfunction of liver and kidneys, which contributes to the understanding of combined toxic effects of the pesticides revealed by using the metabolomics analysis of the urine and serum profiles.

Quantitative bile acid profiling in healthy adult dogs and pups from serum, plasma, urine, and feces using LC-MS/MS.

Synthesis and secretion of bile acids (BA) is a key physiological function of the liver. In pathological conditions like portosystemic shunt, hepatic insufficiency, hepatitis, or cirrhosis BA metabolism and secretion are disturbed. Quantification of total serum BA is an established diagnostic method to assess the general liver function and allows early detection of abnormalities, liver disease progression and guidance of treatment decisions. To date, data on comparative BA profiles in dogs are limited. However, BA profiles might be even better diagnostic parameters than total BA concentrations. On this background, the present study analyzed and compared individual BA profiles in serum, plasma, urine, and feces of 10 healthy pups and 40 adult healthy dogs using ultra-high performance liquid chromatography coupled to electrospray ionization mass spectrometry. Sample preparation was performed by solid-phase extraction for serum, plasma, and urine samples or by protein precipitation with methanol for the feces samples. For each dog, 22 different BA, including unconjugated BA and their glycine and taurine conjugates, were analyzed. In general, there was a great interindividual variation for the concentrations of single BA, mostly exemplified by the fact that cholic acid (CA) was by far the most prominent BA in blood and urine samples of some of the dogs (adults and pups), while in others, CA was under the detection limit. There were no significant age-related differences in the BA profiles, but pups showed generally lower absolute BA concentrations in serum, plasma, and urine. Taurine-conjugated BA were predominant in the serum and plasma of both pups (68%) and adults (74-75%), while unconjugated BA were predominant in the urine and feces of pups (64 and 95%, respectively) and adults (68 and 99%, respectively). The primary BA chenodeoxycholic acid and taurocholic acid and the secondary BA deoxycholic acid and lithocholic acid were the most robust analytes for potential diagnostic purpose. In conclusion, this study reports simultaneous BA profiling in dog serum, plasma, urine, and feces and provides valuable diagnostic data for subsequent clinical studies in dogs with different kinds of liver diseases.

Evaluation of Diuretic Activity of Aqueous and Hydro Methanolic Crude Extracts and Solvent Fraction of the Hydromethanolic Flower Extract of Erica Arborea L. (Ericaceae) in Swiss Albino Mice.

To evaluate the diuretic effects of aqueous (AQ) and hydromethanolic crude extract (HM) the as well as the solvent fractions of the HM extract from Erica arborea flowers in mice. Mice were administered AQ and HM crude extracts, along with solvent fractions of HM extracts of E. arborea flowers, including HXF (n-hexane fraction), EAF (ethyl acetate fraction), and AQF (aqueous fraction), at doses ranging from 100 to 400 mg/kg orally. The effects of these extracts and solvent fractions on urine and salt excretion over 5 hours were compared to the effects of the solvent used for reconstitution and a standard drug (furosemide 10 mg/kg), as well as to each other. The HM crude extract at a lower dose (100 mg/kg) significantly increased urine volume and salt excretion starting from the 3rd h compared to the AQ crude extract. Similar effects were observed for EAF. Notably, the HM extract and its EAF at 400 mg/kg showed comparable urine and salt excretion profiles to the standard drug. This study demonstrated that HM extract and EAF promote better diuresis, likely due to their saluretic properties. Furthermore, it confirms the diuretic activity of Erica arborea flowers.

Chronic consumption of a bergamot-based beverage does not affect glucose, lipid and inflammatory biomarkers of cardiometabolic risk in healthy subjects: a randomised controlled intervention study.

Background: Pure bergamot juice exerts lipid lowering effects in dyslipidemic subjects. It is unknown whether bergamot-based beverages exert similar effects in healthy subjects. Aim: To assess the effects, if any, of a bergamot-based beverage (BBB, bergamot juice ≤25%) on lipid, metabolic and inflammatory biomarkers. Methods: Forty-five healthy subjects were randomised 1 : 1 to BBB intake (400 mL day-1) (55.5%) or control (44.5%) for 12 weeks. Anthropometric (waist circumference, body mass index (BMI)) and clinical (blood pressure) parameters, blood samples (glucose, glycated haemoglobin, insulinemia, lipid profile, liver and renal function, inflammatory biomarkers) and 24-h urine for the analysis of (poly)phenol metabolites were collected at the baseline and at 12 weeks. Intakes of energy, nutrients and food groups were assessed by a 7-day dietary record. Results: Both groups exhibited a time-related significant decrease in total cholesterol (p = 0.02), fasting plasma glucose (p = 0.016), insulin (p = 0.034), BMI (p < 0.001) and waist circumference (p = 0.04), but with no significant between-arm difference. The urinary profile of metabolites from the BBB-derived (poly)phenols well discriminated the two study groups, documenting good compliance in the intervention arm. Notably, urinary bergamot 3-hydroxy-3-methylglutaryl (HMG) -containing flavanones or derived HMG-containing metabolites were not detectable. BBB was well tolerated and no adverse events were recorded. Conclusion: This first randomized controlled trial of BBB consumption in healthy subjects showed no effects of BBB on the cardiometabolic risk profile. BBB consumption is a safe nutritional adjunct in the context of a well balanced diet.

Dryfilm-ATR-FTIR analysis of urinary profiles as a point-of-care tool to evaluate aerobic exercise.

The understanding of metabolic alterations triggered by intense exercise can provide a biological basis for the development of new training and recovery methods. One popular way to monitor these changes is the non-invasive analysis of the composition of urine. This work evaluates the use of attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and multivariate analysis as a rapid and cost-effective way to investigate changes in urine composition after intense exercise. The urine FTIR spectra of 21 volunteers (14 going through aerobic exercise and 7 controls) were measured before and immediately, 2, 5, 11, and 24 h after running 10 km. Principal component analysis (PCA) and partial least squares analysis (PLS) regression were used to investigate the changes in the spectra as a function of the recovery time. PLS models obtained for the prediction of the time points in the exercise group were deemed significant (p < 0.05, rand t-test permutation testing in cross-validation), showing changes in the urine composition after the exercise, reaching a maximum after 11 hours as opposed to the control group which did not show any significant relationship with the recovery time. In a second step, spectra of the protean extract isolated from urines at significant timepoints (before, immediately after, and 11 hours after exercise) were measured. The PCA of the protein spectra showed clear differences in the spectra obtained at the separation between the recovery time points, especially after the end of the exercise, where the protein profile was significantly different from the other times. Results indicate that the technique was able to find differences in the urine after physical exertion and holds strong potential for an easy-to-use and simple screening metabolic evaluation of recovery methods.

Targeting Gut Microbiome With Prebiotic in Patients With CKD: The TarGut-CKD Study

IntroductionDisruption of gut microbiota underpins some of the metabolic alterations observed in chronic kidney disease (CKD). MethodsIn a non-randomized, open-label, 3-phase pilot trial, with repeated measures within each phase, we examined the efficacy of oligofructose-enriched inulin (p-inulin) in changing the gut microbiome and their metabolic products in 15 CKD patients. The stability of microbiome and metabolome was studied during the pre-treatment phase (8 weeks), a p-inulin treatment phase (12 weeks), and a post-treatment phase (8 weeks) of the study. ResultsStudy participants completed 373 of the 420 expected study visits (88.8%). Adherence to p-inulin was 83.4%. 16S rRNA sequencing was performed in 368 stool samples. A total of 1085 stool, urine, and plasma samples were subjected to untargeted metabolomic studies. P-inulin administration altered the composition of the gut microbiota significantly, with an increase in abundance of Bifidobacterium and Anaerostipes. Inter-subject variations in microbiome and metabolome were larger than intra-subject variation, indicating the stability of the gut microbiome within each phase of the study. Overall metabolite compositions assessed by beta diversity in urine and stool metabolic profiles were significantly different across study phases. Several specific metabolites in stool, urine and plasma were significant at FDR ≤ 0.1 over phase. Specifically, there was significant enrichment in microbial metabolites derived from saccharolysis. ConclusionResults from our study highlight the stability of the gut microbiome and the expansive effect of p-inulin on microbiome and host co-metabolism in CKD patients. Findings from this study will enable rigorous design of microbiome-based intervention trials.

Gut microbiome and metabolic profiles of mouse model for MeCP2 duplication syndrome

The extra copy of the methyl-CpG-binding protein 2 (MeCp2) gene causes MeCP2 duplication syndrome (MDS), a neurodevelopmental disorder characterized by intellectual disability and autistic phenotypes. However, the disturbed microbiome and metabolic profiling underlying the autistic-like behavioral deficits of MDS are rarely investigated. Here we aimed to understand the contributions of microbiome disruption and associated metabolic alterations, especially the disturbed neurotransmitters in MDS employing a transgenic mouse model with MeCP2 overexpression. We analyzed metabolic profiles of plasma, urine, and cecum content and microbiome profiles by both 16 s RNA and shotgun metagenomics sequence technology. We found the decreased levels of Firmicutes and increased levels of Bacteroides in the single MeCP2 gene mutation autism-like mouse model, demonstrating the importance of the host genome in a selection of microbiome, leading to the heterogeneity characteristics of microbiome in MDS. Furthermore, the changed levels of several neurotransmitters (such as dopamine, taurine, and glutamate) implied the excitatory-inhibitory imbalance caused by the single gene mutation. Concurrently, a range of microbial metabolisms of aromatic amino acids (such as tryptophan and phenylalanine) were identified in different biological matrices obtained from MeCP2 transgenic mice. Our investigation revealed the importance of genetic variation in accounting for the differences in microbiomes and confirmed the bidirectional regulatory axis of microbiota-gut-brain in studying the role of microbiome on MDS, which could be useful in deeply understanding the microbiome-based treatment in this autistic-like disease.

Integration of lipidomics and metabolomics reveals plasma and urinary profiles associated with pediatric Mycoplasma pneumoniae infections and its severity.

Mycoplasma pneumoniae is a significant contributor to lower respiratory infections in children. However, the lipidomics and metabolics bases of childhood M. pneumoniae infections remain unclear. In this study, lipidomics and metabolomics analyses were conducted using UHPLC-LTQ-Orbitrap XL mass spectrometry and gas chromatography-triple quadrupole mass spectrometry on plasma (n = 65) and urine (n = 65) samples. MS-DIAL software, in combination with LipidBlast and Fiehn BinBase DB, identified 163 lipids and 104 metabolites in plasma samples, as well as 208 metabolites in urine samples. Perturbed lipid species (adjusted p < 0.05) were observed, including lysophosphatidylethanolamines, phosphatidylinositols, phosphatidylcholines, phosphatidylethanol amines, and triglycerides. Additionally, differential metabolites (adjusted p < 0.05) exhibited associations with amino acid metabolism, nucleotide metabolism, and energy metabolism. Thirteen plasma metabolites, namely l-hydroxyproline, 3-phosphoglycerate, citric acid, creatine, inosine, ribitol, α tocopherol, cholesterol, cystine, serine, uric acid, tagatose, and glycine, showed significant associations with disease severity (p < 0.05) and exhibited distinct separation patterns in M. pneumoniae-infected bronchitis and pneumonia, with an area under the curve of 0.927. Nine of them exhibited either positive or negative correlations with neutrophil or lymphocyte percentages. These findings indicated significant systemic metabolic shifts in childhood M. pneumoniae infections, offering valuable insights into the associated metabolic alterations and their relationship with disease severity.

Interaction between a Sulfated Polysaccharide from Sea Cucumber and Gut Microbiota Influences the Fat Metabolism in Rats.

Due to its significant physiological effects, a sulfated polysaccharide has been considered an important nutrient of sea cucumber, but its metabolism in vivo is still unclear. The present study investigated the metabolism of a sea cucumber sulfated polysaccharide (SCSP) in rats and its influence on the metabolite profiles. The quantification by HPLC-MS/MS revealed that the blood level of SCSP achieved a maximum of 54.0 ± 4.8 μg/mL at 2 h after gavage, almost no SCSP was excreted through urine, and 55.4 ± 29.8% of SCSP was eliminated through feces within 24 h. These results prove the utilization of SCSP by gut microbiota, and a further microbiota sequencing analysis indicated that the SCSP utilization in the gut was positively correlated with Muribaculaceae and Clostridia_UCG-014. In addition, the non-targeted metabolomic analysis demonstrated the significant effects of SCSP administration on the metabolite profiles of blood, urine, and feces. It is worth noting that the SCSP supplement decreased palmitic acid, stearic acid, and oleic acid in blood and urine while increasing stearic acid, linoleic acid, and γ-linolenic acid in feces, suggesting the inhibition of fat absorption and the enhancement of fat excretion by SCSP, respectively. The present study shed light on the metabolism in vivo and the influence on the fat metabolism of SCSP.